Marshall UniversityMarshall Digital Scholar

Biochemistry and Microbiology Faculty Research

Fall 11-14-2016

Epoxyeicosatrienoic acids regulate adipocytedifferentiation of mouse 3T3 cells, via PGC-1αactivation, which is required for HO-1 expressionand increased mitochondrial functionMaayan Waldman

Lars Bellner

Luca Vanella

Joseph Schragenheim

Komal SodhiMarshall University, sodhi@marshall.edu

See next page for additional authors

Follow this and additional works at: http://mds.marshall.edu/sm_bm

Part of the Medical Biochemistry Commons, and the Other Medical Sciences Commons

This Article is brought to you for free and open access by the Faculty Research at Marshall Digital Scholar. It has been accepted for inclusion inBiochemistry and Microbiology by an authorized administrator of Marshall Digital Scholar. For more information, please contactzhangj@marshall.edu, martj@marshall.edu.

Recommended CitationWaldman M, Bellner L, Vanella L, Schragenheim J, Sodhi K, Singh SP, Lin D, Lakhkar A, Li J, Hochhauser E, Arad M.Epoxyeicosatrienoic acids regulate adipocyte differentiation of mouse 3T3 cells, via PGC-1α activation, which is required for HO-1expression and increased mitochondrial function. Stem Cells and Development. 2016;25:1084-1094. http://online.liebertpub.com/doi/pdfplus/10.1089/scd.2016.0072

AuthorsMaayan Waldman, Lars Bellner, Luca Vanella, Joseph Schragenheim, Komal Sodhi, Shailendra P. Singh,Daohong Lin, Anand Lakhkar, Jiangwei Li, Edith Hochhauser, Michael Arad, Zbigniew Darzynkiewicz,Attallah Kappas, and Nader G. Abraham

This article is available at Marshall Digital Scholar: http://mds.marshall.edu/sm_bm/200

Epoxyeicosatrienoic Acids Regulate AdipocyteDifferentiation of Mouse 3T3 Cells, Via PGC-1a Activation,

Which Is Required for HO-1 Expression and IncreasedMitochondrial Function

Maayan Waldman,1,2,* Lars Bellner,1,* Luca Vanella,1,3,* Joseph Schragenheim,1 Komal Sodhi,4

Shailendra P. Singh,1 Daohong Lin,1 Anand Lakhkar,1 Jiangwei Li,5 Edith Hochhauser,2 Michael Arad,6

Zbigniew Darzynkiewicz,5 Attallah Kappas,7 and Nader G. Abraham1,4,7,8

Epoxyeicosatrienoic acid (EET) contributes to browning of white adipose stem cells to ameliorate obesity/diabetes and insulin resistance. In the current study, we show that EET altered preadipocyte function, enhancedperoxisome proliferation-activated receptor g coactivator a (PGC-1a) expression, and increased mitochondrialfunction in the 3T3-L1 preadipocyte subjected to adipogenesis. Cells treated with EET resulted in an increase,P < 0.05, in PGC-1a and a decrease in mitochondria-derived ROS (MitoSox), P < 0.05. The EET increase inheme oxygenase-1 (HO-1) levels is dependent on activation of PGC-1a as cells deficient in PGC-1a (PGC-1aknockout adipocyte cell) have an impaired ability to express HO-1, P < 0.02. Additionally, adipocytes treatedwith EET exhibited an increase in mitochondrial superoxide dismutase (SOD) in a PGC-1a-dependent manner,P < 0.05. The increase in PGC-1a was associated with an increase in b-catenin, P < 0.05, adiponectin expres-sion, P < 0.05, and lipid accumulation, P < 0.02. EET decreased heme levels and mitochondria-derived ROS(MitoSox), P < 0.05, compared to adipocytes that were untreated. EET also decreased mesoderm-specifictranscript (MEST) mRNA and protein levels (P < 0.05). Adipocyte secretion of EET act in an autocrine/paracrine manner to increase PGC-1a is required for activation of HO-1 expression. This is the first study todissect the mechanism by which the antiadipogenic and anti-inflammatory lipid, EET, induces the PGC-1asignaling cascade and reprograms the adipocyte phenotype by regulating mitochondrial function and HO-1expression, leading to an increase in healthy, that is, small, adipocytes and a decrease in adipocyte enlargementand terminal differentiation. This is manifested by an increase in mitochondrial function and an increase in thecanonical Wnt signaling cascade during adipocyte proliferation and terminal differentiation.

Introduction

The cytochrome P450 (CYP) monooxygenase/epoxygenasefamily is responsible for formation of 20-hydroxyeico-

satetraenoic acid (20-HETE) and epoxyeicosatrienoic acids(EETs) [1,2]. Upon formation, EETs are rapidly hydrolyzedby soluble epoxide hydrolases (sEHs) and reactive oxygenspecies (ROS) [heme oxygenase-1 (preventable by HO-1)induction] to their respective dihydroxyepoxytrienoic acids(DHETs), as well as to esterification primarily to glycero-phospholipids. Vasodilatory, anti-inflammatory, and antiapop-

totic actions of EETs are well established, and sEH inhibitionsignificantly increases cellular and circulating EET levels [3,4].EET increases osteoblast differentiation but decreases adipocytedifferentiation via activation of HO-1 (HO-1) [5–7]. In 3T3adipocyte cells, an increase in heme increases adipogenesis[8,9], which is associated with a glucose-mediated increasein ROS, which inactivates HO-1 (reviewed [10]).

The 3T3 adipocyte is a model to study the role of PGC-1as thermogenic activators, the mitochondrial heme path-way, and biogenesis [11] as well as the evaluation of hemehomeostasis [12]. However, an increase in HO-1 gene

Departments of 1Pharmacology, 5Pathology, and 8Medicine, New York Medical College, Valhalla, New York.2Cardiac Research Laboratory, Felsenstein Medical Research Institute, Tel-Aviv University, Petah-Tikva, Israel.3University of Catania, Department of Drug Science/Section of Biochemistry, Catania, Italy.4Departments of Medicine and Surgery, Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia.6Leviev Heart Center, Sheba Medical Center, Tel Hashomer and Sackler School of Medicine, Tel Aviv University, Tel Hashomer, Israel.7The Rockefeller University, New York, New York.*These authors contributed equally to this work.

� Maayan Waldman, et al., 2016; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms ofthe Creative Commons Attribution Noncommercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits any non-commercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

STEM CELLS AND DEVELOPMENT

Volume 25, Number 14, 2016

Mary Ann Liebert, Inc.

DOI: 10.1089/scd.2016.0072

1084

expression decreases adipocyte heme and subsequentlyattenuates (hypertrophy) adipogenesis [10]. This reductionin adipogenesis is associated with an increase in the levelsof EETs [13], which increase hematopoietic stem celltransplant engraftment that is associated with activation ofthe transcriptional factor activator protein 1 (AP-1) [14].The AP-1 binding site is present in the human HO-1 pro-moter that activates HO-1 gene expression [15]. Therefore,the mechanism by which EET increases HO-1 expressionmay not be related solely to inhibition by the BTB and CNChomology 1, basic leucine zipper transcription factor 1 (Bach1) [16]. EET affects adipocyte differentiation and hypertro-phy both in vitro and in vivo [7,17,18] by reprogramming theadipocyte phenotype.

The development of adipocytes in mice and humansfollows a clearly defined pathway (adipogenesis) that beginswith a common mesenchymal stem cell (MSC) that is plu-ripotent [19]. The early steps of the pathway leading to thegeneration and the commitment of MSCs to adipocyte lin-eage are unknown. Hypothetically, the determination of thefate of MSCs occurs early in the stages of cell differentia-tion (‘‘commitment’’) and involves the interplay of intrinsic(genetic) and environmental (local and systemic) conditionsto ultimately define cell fate. Factors such as those discussedearlier, that determine MSC proliferation and differentia-tion, also govern early adipocyte development and function.Currently, little is known about this process—from MSC topreadipocytes to adipocyte differentiation.

During adipogenesis, MSCs or preadipocytes differentiateinto lipid-laden and insulin-sensitive adipocytes [20]. Theacquisition of adipocyte phenotype and development of adi-pocyte function is characterized by chronological changes inthe expression of multiple genes. Briefly, the stages of adi-pocyte differentiation are as follows: (1) MSC-derived adi-pocyte growth arrest, (2) clonal expansion, (3) a second stageof growth arrest or early differentiation, and (4) terminaldifferentiation—development of a mature adipocyte pheno-type [21,22]. Concurrently as cells proceed to differentiation,there is an increase in transcription or de novo expression ofseveral genes, including adiponectin, Glut 4, insulin receptor,and fatty acid synthase [21]. In the early phase of differen-tiation, preadipocyte cells are morphologically similar to fi-broblasts. After clonal expansion, continued induction ofadipogenesis leads to a drastic change in cell shape. Pre-adipocytes convert to a spherical shape; lipid droplets accu-mulate, and the preadipocyte progressively acquires themorphological and biochemical characteristics of a matureadipocyte, followed by triglyceride accumulation [22].

Administration of EET or inhibitors of sEH to obese miceis associated with a decrease in visceral subcutaneous fat andan increase in insulin sensitivity [23–26]. The CYP2J2-mediated increase in EET increases fatty acid oxidation andadiposity [17,27,28]. Although EETs exhibit potent biologi-cal effects, including vasodilatation and inhibition of the in-flammatory response [4,29,30] and in adiposity [17,27,28],their influence on mitochondrial function and proliferator-activated receptor gamma coactivator-1a (PGC-1a) in rela-tion to adipogenesis remains unknown.

Peroxisome PGC-1a was originally described as a coac-tivator of peroxisome proliferator-activated receptor-gamma(PPAR-g) that modulates expression of uncoupling protein 1(UCP-1) and thermogenesis in brown fat [31,32]. PGC-1a

controls mitochondrial biogenesis and oxidative metabolismin many tissues, including brown adipose tissue [11,33].Transgenic mice with mildly elevated muscle PGC-1a areresistant to age-related obesity and diabetes resulting in aprolongation of their life span, suggesting that PGC-1a in-fluences the secretion of factors of biological influence thataffect the function of other tissues [34]. Adipose-specificPGC-1a deficiency results in a decrease in mitochondrialbiogenesis and an increase in fatty acid oxidation, glucose,and insulin resistance [35].

In the present study, we examined whether the EET-mediated regulation of adiposity is due to activation ofPGC-1a and an increase in UCP-1, HO-1, and b-catenin,resulting in decreased adipocyte terminal differentiationthrough the regulation of heme levels, essential for adipo-cyte proliferation and differentiation.

Materials and Methods

Cell culture

3T3-L1 mouse preadipocytes were purchased from theATCC (ATCC, Manassas, VA). After thawing, 3T3-L1cells were resuspended in an a-minimal essential medium(a-MEM; Invitrogen, Carlsbad, CA) supplemented with 10%heat-inactivated fetal bovine serum (FBS; Invitrogen) and1% antibiotic/antimycotic solution (Invitrogen). The cul-tures were maintained at 37�C in a 5% CO2 incubator, andthe medium was changed after 48 h and every 3–4 daysthereafter. When the 3T3-L1 cells were confluent, the cellswere recovered by the addition of 0.25% trypsin/EDTA(Invitrogen) and subcultured. For experiments, 3T3-L1 cellswere seeded in 96- and 24-well plates at a density of 10,000cells/cm2 and cultured in a-MEM with 10% FBS until 90%confluence was achieved. The medium was then replacedwith adipogenic medium, and the cells were cultured foran additional 7 days. The adipogenic medium consisted ofDulbecco’s modified Eagle’s medium (DMEM) with highglucose (Invitrogen) supplemented with 10% (v/v) FBS,10mg/mL insulin (Sigma-Aldrich, St. Louis, MO), 0.5 mMdexamethasone (Sigma-Aldrich), and 0.1 mM indomethacin(Sigma-Aldrich). For some experiments, cells were treatedwith CoPP (Cobalt protoporphyrin; Frontier Scientific, Lo-gan, UT) at a final concentration of 2–5mM, as indicated inthe figure legends; for 5–7 days, the EET analog, EET-A[(S)-2-(11-(nonyloxy) undec-8(Z)-enamido) succinic acid],was treated at a final dose of 1–10 mM, as indicated in thefigure legends. To examine the effect of inhibition of HOactivity on EET-A-treated cell, SnMP (2 mM, Tin meso-porphyrin; Frontier Scientific) was added for the duration ofEET-A treatment. Cells and conditioned media were col-lected after 7 days, as indicated in the figure legends.

At the experimental endpoints, cells were collected bytrypsinization, washed once with PBS, and then lysed forboth protein measurement and RNA extraction.

Isolation and development of PGC-1a-deficientadipocytes

SMART vector lentiviral shRNA-PPARGC1A or scram-bled RNA (Dharmacon, Lafayette, CO) was applied to 3T3-L1cells to establish a stably transduced cell line. Briefly, 1 · 106

cells were seeded in six-well plates 1 day before transduction.

EET UPSTREAM OF PGC-1a REGULATES ADIPOCYTE DEVELOPMENT 1085

On the day of transduction, the transduction medium wasmade by 1 · 106 transducing units of lentiviral particles with0.5 mL a-MEM growth medium applied to each well and in-cubated for 3 h to maximize the contact between each cell andlentiviral particles. Cells were also treated with the transduc-tion medium without lentiviral particles, which served as un-transduced control. Growth medium (1.5 mL) was then addedto each well in the presence of 8mg/mL polybrene (finalconcentration). After 48-h incubation, antibiotic selectionmedium (a-MEM growth medium with 10mg/mL puromycin)was used to kill all the untransduced cells for either lenti-scramble or cells treated with lentiviral shRNA-PPARGC1A.The growth medium was replaced every 2 days and used fordifferentiation and experimental studies.

Oil Red O staining

0.21% Oil Red O in 100% isopropanol (Sigma-Aldrich)was used for staining, as previously described [18]. Briefly,adipocytes were fixed in 10% formaldehyde, stained with OilRed O for 10 min, and rinsed with 60% isopropanol (Sigma-Aldrich), where after the Oil Red O was eluted by incubationin 100ml 100% isopropanol for 10 min and then the OD readat 490 nm for 0.5 s, as previously described [7,18].

Lipid droplet size analysis. The lipid droplet size andnumber were acquired by analyzing the whole bottom surfacearea of an individual well of a 96-well plate. Each experimentwas performed in triplicate, and the droplet analysis wasperformed [G]. The photographs are representative images ofthe experimental conditions. Cell size was measured using anImagePro Analyzer (MediaCybernetics, Inc., Rockville,MD). The classification of the size of lipid droplets was basedon the size of the two-dimensional area and expressed aspixels as we previously described [6].

Flow cytometer measurement of MitoSox

Mitochondrial superoxide formation was determined bythe uptake of MitoSox� Red reagent (Invitrogen) in livecells, as previously described [36]. Briefly, cells were platedat a density of 10,000 cells/cm2 in six-well plates, with orwithout EET-A, 10 mM, overnight and then incubated withHoechst nuclear stain (5mg/mL) for 30 min in complete me-dium. The medium was removed, and the cells were incu-bated with MitoSox Red reagent (5 mM) in Hank’s balancedsalt solution for 10 min at 37�C protected from light. The cellswere then washed twice with PBS, collected by trypsiniza-tion, and finally washed with PBS supplemented with 2%bovine serum albumin (BSA; Fisher Scientific, Pittsburg,PA). The cells were then fixed in 2% formaldehyde solutionfor 10 min, collected by centrifugation, and resuspended inPBS supplemented with 2% BSA. Intensity of cellular fluo-rescence was measured using a Beckman Coulter’s MoFloXDP flow cytometer (Beckman Coulter, Indianapolis, IA)and analyzed with Kaluza v1.3 software (Beckman Coulter).More than 16,000 cells were measured per sample. Data arepresented as a frequency distribution graph of fluorescenceintensity of individual cells plotted on a logarithmic coordi-nate; the mean and SD are shown per individual sample. Themean values from triplicate cultures were used to calculatestatistical difference between the control and EET-A-treatedcultures.

HPLC measurement of mitochondria-derivedROS and heme measurement

Mitochondrial superoxide formation was determined bythe uptake of MitoSox Red reagent (Invitrogen) in live cells,as previously described [37,38]. Briefly, cells were plated ata density of 10,000 cells/cm2 in six-well plates with orwithout EET-A, 10 mM, overnight and then incubated withthe MitoSox Red reagent (5mM) in Hank’s balanced saltsolution for 10 min at 37�C protected from light. The cellswere washed twice with PBS, and 300mL acetonitrile wasthen added per well, followed by incubation on ice for 5 minprotected from light. Cells were collected by scraping, andthe lysate was transferred into microcentrifuge tubes. Sam-ples were incubated at -20�C for 1 h, then pelleted bycentrifugation at 14,000g for 10 min, and used for proteinmeasurement, and the supernatant was then collected andstored at -25�C until analyzed for MitoSox fluorescence(excitation/emission 510/580) using an HPLC system with aJasco FP-1520 fluorescence detector and a Beckman ultra-sphere reverse column (C18) (5m, 250 · 4.6 mm), and thevalues obtained were normalized to protein levels. Freeheme and degraded heme protein, including myoglobin,were measured using a BioVision ELISA Kit (BioVision,Inc., Milpitas, CA).

Cytokine measurements

Adiponectin (high molecular weight, HMW), interleukin6 (IL-6), and IL-1a were determined in conditioned mediumusing an ELISA Assay ELISA Kit (R&D Systems, Inc.,Minneapolis, MN) according to the manufacturer’s in-structions and as previously described [7,39].

Immunoblotting of signaling proteins

Cells were trypsinized, pelleted by centrifugation, andlysed with lysis buffer supplemented with protease andphosphatase inhibitors (complete� Mini and PhosSTOP�;Roche Diagnostics, Indianapolis, IA). Protein levels werequantified using a commercial assay (Bio-Rad, Hercules,CA). Protein samples were applied to sodium dodecyl sul-fate (SDS) polyacrylamide gel (10%–15%), electrophoresedunder denaturing conditions, and electrotransferred ontoPVDF Immobilon-P membrane (Amersham Pharmacia,Piscataway, NJ) using a semidry transfer apparatus (Bio-Rad). Membranes were blocked with Odyssey� BlockingBuffer (TBS) (LI-COR, Lincoln, NE) for 1 h at roomtemperature. Primary antibodies (1:500–1:1,000 dilution)in blocking buffer supplemented with 0.1% Tween 20(Fisher Scientific) were incubated overnight at 4�C,washed in Tris-buffered saline (TBS) supplemented with0.1% Tween 20, and then incubated with the appropriatefluorophore-conjugated secondary antibodies (1:5,000–1;20,000) (LI-COR). Detection was completed with a LI-COROdyssey instrument (LI-COR), and quantification of signalswas completed with the Odyssey program (LI-COR).

Quantitative real-time PCR

Briefly, total RNA was extracted from pelleted cells usingTRIzol� (Ambion, Austin, TX) according to the manufacturer’s

1086 WALDMAN ET AL.

instructions. Concentration of total RNA was determined bythe use of a Take3� plate and a Biotek� Plate Reader (Biotek,Winooski, VT). cDNA was synthesized from 1mg total RNAand 18s using the High-Capacity cDNA Reverse Transcrip-tion Kit (Applied Biosystems, Foster City, CA) according tothe manufacturer’s protocol, and PCR mRNA calculation wasadjusted to 18s as control. Quantitative real-time PCR anal-ysis was performed using the 7500 Fast Real-Time PCRSystem (Applied Biosystems). The gene expression analysislevels were determined using the relative expression methodwith the threshold crossing point (Ct-value) as basis and asdescribed [40].

Statistical analysis

The treatment groups were compared with the controlgroup, and the experiments were analyzed using one-wayanalyses of variance (ANOVA) with Bonferroni tests formultiple comparisons. Data are presented as mean – SE. Sta-tistical significance was defined as P < 0.05.

Results

EET reduces adipogenesis in differentiatedadipocytes

We examined the effect of CoPP on HO-1 induction andEET-A on adipocyte function by measuring the total amountof lipids. Differentiated adipocyte cells treated with eitherEET-A or CoPP display reduced large lipid droplet contentcompared with differentiated control cells (P < 0.05), aneffect that was completely prevented by concomitant treat-ment with EET-A and an inhibitor of HO activity, SnMP(Fig. 1A). The results are quantitated in Fig. 1B. The effectof EET-A on adipogenesis was determined by counting cellswith small lipid droplets in the cytoplasm and comparedwith large adipocytes (Fig. 1B). The percentage of cells withsmall lipid droplets increased in cells treated with EET. Indifferentiated cells treated with either EET or EET-SnMPand compared with untreated cells, the percentage of smalland large lipid droplets is displayed in Fig. 1B, left and rightpanels, respectively. EET-A decreased adipocyte terminal

FIG. 1. EET-A and CoPP increasesmall adipocyte number and inhibitadipocyte terminal differentiation.3T3-L1 cells cultured in differenti-ation medium were stained with OilRed O. (A) Representative images ofdifferentiated control cells and cellstreated with EET-A (1mM), EET-A(1mM)+SnMP (5mM), and CoPP(2mM). Analysis of lipid droplet size(B). *P < 0.05 versus control. (C)Representative blot and densitome-try analysis of PGC-1a protein levelsin the presence of EET (1mM) withor without the addition of SnMP(5mM). *P < 0.05 versus control,n = 3. (D) PGC-1a mRNA levels inthe presence of EET-A (10mM)with or without the addition ofSnMP (5mM). *P < 0.05 versus con-trol, n =3. EET-A, epoxyeicosatrienoicacid; PGC-1a, proliferation-activatedreceptor g coactivator a.

EET UPSTREAM OF PGC-1a REGULATES ADIPOCYTE DEVELOPMENT 1087

differentiation, that is, prevented the conversion of smalladipocytes to large adipocytes (Fig. 1B). The adipocyte cellsize in the absence of the EET-A agonist was 180 – 3 pixelscompared with 100 – 2.1 pixels in the presence of EET-A(Fig. 1B, right panel).

We examined the levels of PGC-1a in adipocytes treatedwith EET-A or SnMP. As seen in Fig. 1C, EET-A increasedPGC-1a levels, while SnMP largely prevented this effect.These results were confirmed by the levels of PGC-1amRNA levels (Fig. 1D).

EET decreases inflammation during differentiationof adipocytes

Treatment with EET-A reduced the levels of both IL-1aand IL-6 compared with control, and SnMP blocked thiseffect (Fig. 2A, B). Adiponectin levels in the conditionedmedium of adipocytes treated with EET-A were higher(P < 0.05) compared with untreated adipocytes. SnMPblocked the effect of EET-A (P < 0.05) (Fig. 2C).

HO-1 expression is impaired in adipocytesdeficient in PGC-1a

The expression of PGC-1a protein in the PGC-1a KDcells was 30%–35% of PGC-1a protein levels in WT cells(Fig. 3A, B). Since EET increased PGC-1a levels (Fig. 2)and EET is known to increase HO-1 protein expression, wemeasured HO-1 in WT, Mock, and PGC-1a KD adipocytecells treated with EET-A. In WT and Mock adipocyte cells,EET increased HO-1 levels (Fig. 3C). However, in PGC-1aKD adipocyte cells, EET failed to increase the expression ofHO-1, suggesting that PGC-1a is necessary for HO-1 ex-pression (Fig. 3D).

PGC-1a knockdown decreasesmitochondrial viability

Mitochondrial superoxide levels were elevated in PGC-1a-deficient cells compared with WT cells (Fig. 4A, B), andEET-A significantly reduced mitochondrial superoxidelevels in WT cells (P < 0.05) (Fig. 4C). UCP-1 protein levelswere lower in PGC-1a-deficient cells compared with thosein WT cells (Fig. 4D). Treatment with EET-A increasedUCP-1 protein levels in WT cells (P < 0.05) but did notaffect the UCP-1 levels in PGC-1a-deficient cells (Fig. 4D).Adipocyte cell heme levels were higher in the WT controlcompared with the EET-A-treated cells (Fig. 4E).

PGC-1a knockdown prevents EET-mediatedHO-1 induction

To examine if EET induction of HO-1 is mediatedthrough PGC-1a, preadipocytes were transfected with PGC-1a shRNA. As seen in Fig. 5A and B, the EET-mediatedincrease in HO-1 protein was abolished in PGC-1a-deficientcells, suggesting that EET-A increases HO-1 through thedirect effect of PGC-1a. As shown in Fig. 5C and D, thelevels of manganese superoxide dismutase (MnSOD) arethreefold lower in PGC-1a-deficient adipocyte cells com-pared with WT cells. Moreover, cells treated with EET-Adisplayed an increased level of MnSOD protein in both WTcells and PGC-1a-deficient cells (Fig. 5C, D).

Effect of EET-A on b-catenin and Wnt10b

We examined whether expression of b-catenin andwingless-type MMTV integration site family, member 10b(Wnt10b) is dependent on the levels of PGC-1 expression by

FIG. 2. Inflammatory and adipo-genesis markers. The proinflammatorycytokines IL-1a (A) and IL-6 (B)were measured by ELISA in theconditioned medium of treated andnontreated adipocytes at 7 days ofdifferentiation and treated withEET-A and SnMP. (C) Adiponectin(HMW) levels in differentiated 3T3cells treated with EET-A (1mM).Adiponectin levels secreted into theconditioned medium were measuredusing ELISA, n = 3–5 in each group,*P < 0.05, #P < 0.05, and **P <0.05 versus EET-A (1mM). EET-A,epoxyeicosatrienoic acid; IL-6, inter-leukin 6.

1088 WALDMAN ET AL.

EET. Treatment of adipocyte cells with EET-A significantly(*P < 0.05) increased both b-catenin and Wnt10b mRNAexpression levels compared with control (Fig. 6A, B), sug-gesting the involvement of HO-1-PGC-1 signaling in regu-lation of stage of differentiations of mouse adipocytes.

Effect of EET-A on MEST mRNA and protein levels

To further characterize the effect of EET-A on adipocytesduring maturation, we examined the levels of mesoderm-specific transcript/paternally expressed gene 1 protein(MEST/Peg1). As seen in Fig. 7A and B, treatment of ad-ipocyte cells with EET-A (10mM) during differentiation for7–8 days reduced MEST mRNA and protein levels com-pared with vehicle control cells.

Discussion

This is the first study to demonstrate that EET, a lipidmediator, is situated upstream of the PGC-1a signalingcascade and plays a critical role in the regulation of adi-pocyte differentiation. Furthermore, this process involves anincrease in HO-1 expression and inhibition of the progres-sion toward terminal adipocyte differentiation. These novelfindings highlighting EET–PGC-1a interplay suggest thatthis plays a significant role in adipocyte proliferation, clonalexpansion, growth arrest, and differentiation. The tran-scriptional coactivator PGC-1a, first identified as a regulator

of PPAR-g in brown adipose tissue rich in the mitochondria,has a special role in thermogenesis [31]. PGC-1a is highlyexpressed in tissues with a high energy demand, such as theheart, skeletal muscle, and brown adipose tissue, where itplays a critical role in maintaining mitochondrial biogenesisand function and as well as cellular energy metabolism[11,41]. In PGC-1a-deficient cells, HO-1 levels are reducedcompared with wild-type cells or Mock cells (Fig. 3C, D).This novel finding adds support to our report that PGC-1a isessential for the EET-mediated increase in HO-1 expressionand a critical role for PGC-1a in reducing adipogenesis bycontrolling heme levels. These results identify PGC-1a as akey component of the regulatory pathway involved in thecontrol of mitochondria-derived ROS and adiponectin re-lease. PGC-1a expression is increased in response to phys-iological stimuli that demand increased mitochondrialenergy production, such as exercise and fasting [42–45], andmay play an important role in the development of antiobe-sity therapies.

Exogenous EET reduced clonal expansion of adipocytes,thereby favoring healthy small adipocytes, as opposed to hy-pertrophic lipid accumulation. Protection against adipose tis-sue dysfunction, including preservation of anti-inflammatoryadipokines such as adiponectin, is related to an adipocyte ofsmaller size [7,10,23,46]. The beneficial effect of EET-A wasblocked by SnMP, indicating that EET is working through theHO system, which controls the cellular levels of heme, leading

FIG. 3. (A and B) PGC-1a protein isreduced in the 3T3-L1 preadipocyte.Data are shown as mean band densitynormalized to b-actin (mean – SE;n = 3; *P < 0.05 compared to WT). (Cand D) The effect of EET on HO-1expression in WT and PGC-1a KDadipocyte cells was compared. Fullydifferentiated cells were treated with1 mM EET-A for 2 days in WT, Mock,and PGC-1a KD cells. Cells werelysed, and protein expression was de-termined (*P < 0.01 compared to WT,n = 3). EET-A, epoxyeicosatrienoicacid; PGC-1a, proliferation-activatedreceptor g coactivator a.

EET UPSTREAM OF PGC-1a REGULATES ADIPOCYTE DEVELOPMENT 1089

FIG. 4. (A) Representative plotshowing flow cytometry analysis ofmitochondrial superoxide levels in WT(left panel) and PGC-1a-deficient (rightpanel) undifferentiated adipocytes and(B) statistical analysis of the integratedMitoSox� fluorescence intensity, n = 3,*P < 0.05 compared to WT. (C) Mi-tochondrial superoxide levels in WTadipocytes as measured by MitoSoxincorporation and analysis of HPLC,n = 3, *P < 0.05 versus control. (D)Representative western blot showingUCP-1 protein levels in WT and PGC-1a-deficient cells (top) and the densi-tometry analysis (bottom), n = 3, *P <0.05 compared to control. (E) Mea-surement of adipocyte heme levels,n = 3, *P < 0.05 compared to control.PGC-1a, proliferation-activated re-ceptor g coactivator a; UCP-1, un-coupling protein 1.

FIG. 5. Western blot analysis for the expression of the HO-1 protein in WT and PGC-1a-deficient cells with and withoutEET-A (10mM) treatment. Representative blots of HO-1 and b-actin (A) and densitometry analysis of HO-1 protein levels,normalized to b-actin in untreated (control) and cells treated for 7 days with EET-A, added every other day (B). Westernblot analysis of the expression of the antioxidant protein MnSOD in differentiated adipocytes. WT- and PGC-1a-silencedcells with and without EET-A (10mM) treatment. Representative blots of MnSOD and b-actin (C) and densitometryanalysis of MnSOD protein levels, normalized to b-actin in untreated (control) and cells treated for 7 days or 16 h after EET-A supplementation for protein measurement and mRNA, respectively, following treatment with EET-A (D). n = 3, *P < 0.05compared to WT; **P < 0.01 compared to WT EET-A treated. EET-A, epoxyeicosatrienoic acid; HO-1, heme oxygenase-1;MnSOD, manganese superoxide dismutase; PGC-1a, proliferation-activated receptor g coactivator a.

1090

to terminal adipocyte stem cell differentiation and enlarge-ment. It is clear that PGC-1a regulates heme levels by twomechanisms: by increased 5¢-aminolevulinate synthase(ALAS, the rate-limiting enzyme of heme biosynthesis) andheme levels needed for full differentiation; simultaneously,PGC-1a increases HO-1 as a result of EET treatment thatreduces heme levels and prevents terminal adipocyte differ-entiation. SnMP increases levels of cellular heme by inhibitingheme oxygenase, the rate-limiting enzyme in heme degrada-tion [47]. We report here that EET increases PGC-1a, an effectnot blocked by SnMP. This confirms previous studies showingthat PGC-1a is particularly sensitive to intracellular hemelevels, particularly via Rev-Erba regulation [12], and plays asignificant role in the increase in the levels of heme by upre-gulation of ALAS [48]. However, EET-mediated increase inPGC-1a leading to increased levels of HO-1 also reducedheme to control adipocyte cells during terminal differentia-tion. Interestingly, SnMP increases cellular heme levels, whileEET increases HO-1 via PGC-1a. Therefore, the net effect ofSnMP treatment did not negatively impact PGC-1a mRNA orprotein levels when added to cells together with EET. This is inall probability due to the fact that EET decreases heme andSnMP increases heme, leaving PGC-1a levels unchanged. Wespeculate that the EET-mediated increase in PGC-1a affectsadipocyte expansion and full differentiation by several

mechanisms: (1) by acting upstream of PGC-1a and by de-creasing heme levels via induction of HO-1, (2) by increasingmitochondrial MnSOD and UCP-1, and (3) through a decreasein MEST/PEG1, necessary for terminal differentiation andadipocyte enlargement [7,13,18,23,28,49–52].

FIG. 6. Effect of EET-A (10mM) on expression ofb-cateninand Wnt10b, mRNA, and/or protein levels. (A) b-cateninmRNA levels. (B) Wnt10b mRNA levels, n = 3, *P < 0.05versus control. EET-A, epoxyeicosatrienoic acid; Wnt10b,wingless-type MMTV integration site family, member 10b.

FIG. 7. Effect of EET-A (10mM) on MEST mRNA (A) andprotein (B) levels after 7–8 days of differentiation. n = 3,*P < 0.05 versus control. MEST, mesoderm-specific transcript.

FIG. 8. Schematic presentation of the possible mechanism bywhich EET-PGC-1a modulates pluripotent hematopoietic stemcell precursors that give rise to a mesenchymal precursor cellwith the potential to differentiate along mesodermal lineagesinto myoblast, including adipocytes. When MSC is placed underappropriate adipogenic conditions, MSC begins to exhibit spe-cific gene expression in preadipocytes that undergo prolifera-tion, clonal expansion, and growth arrest. EET synthesis byCyp2c44 increases PGC-1a levels and translocation to the nu-cleus. In the nucleus together with PGC-1a induced the ex-pression of HO-1 and UCP-1. HO-1 reduces mitochondrialsuperoxide [55] and increases Cyp2c44 levels and activity. Ar-rows indicate that PGC-1a-HO-1 effect is present at 2–3 steps inthe differentiation of MSC to terminally differentiated adipo-cytes. During this process, PGC-1a increases HO-1 expression,which is associated with a decrease in mitochondria-derivedROS (MitoSox). HO-1 gene activation resulted in reprogram-ming the adipocyte phenotype to express a new healthy phe-notype, as evidenced by the expression of fatty acid oxidationenzymes, Wnt10b signaling, and release of adiponectin, whilePGC-1a increases the expression of thermogenic genes, in-cluding UCP-1, decreases MEST, and prevents cells enteringinto proinflammatory cytokines, increasing detrimental sys-temic effects on other organs. EET, epoxyeicosatrienoic acid;HO-1, heme oxygenase-1; MSC, mesenchymal stem cell; PGC-1a, proliferation-activated receptor g coactivator a; ROS, re-active oxygen species; UCP-1, uncoupling protein 1; Mfn1,Mfn2, mitochondrial fusion proteins-1 and 2.

EET UPSTREAM OF PGC-1a REGULATES ADIPOCYTE DEVELOPMENT 1091

HO-1 has multiple cellular effects, including regulatingbioavailability of heme and heme-containing denaturedproteins [53], generation of bioactive metabolites—carbonmonoxide (CO) and biliverdin (BV), preventing the cellularbuildup of free (nonprotein bound) heme, and preventingfree radical and mitochondrial dysfunction [53,54]. HO-1plays a role in mitochondrial biogenesis and function andcytochrome oxidase [55,56]. The effect of PGC-1a on mi-tochondrial function and adipogenesis is exemplified by theEET-A-mediated increase in Cyp2c44, leading to a newtranscriptional role during adipogenesis, for example, byactivation of PGC-1a expression and reduction in mito-chondrial ROS levels (Fig. 4). These results provide a newpathway comprising EET, PGC-1a, and HO-1 that forms apositive feedback loop that stimulates mitochondrial func-tion. Thus, EET, PGC-1a, and HO-1 appear to form amodule that serves as a molecular ‘‘switch’’ to geneticallyreprogram the adipocyte phenotype to express lower levelsof MEST (Fig. 7A, B) and prevent hypoadiponectinemia[7,18] through upregulation of PGC-1a and an increase inmitochondrial function and a decrease in MitoSox.

EET is the first lipid mediator derived from lipid metabolismvia the CYP system to regulate PGC-1a. A dearth of EET isconsidered to be the leading promoter of adipocyte hypertro-phy and dysfunction (large adipocyte). Cells treated with EETare associated with upregulation of PGC-1a-HO-1, suggestingthat EETs regulate adipocyte progression toward differentia-tion and adipogenesis through the activation of PGC-1a sig-naling. Moreover, the protein level of the adipogenic MESTincreased with differentiation (data not shown). Importantly,the MEST mRNA and protein levels were reduced by treat-ment of cells with EET-A during differentiation (Fig. 7).

We show here a direct link between EET-mediated activa-tion of PGC-1a and reduction in mitochondrial ROS produc-tion results in an increase in the number of small adipocytes.This novel finding demonstrates that EET is associated withepigenetic changes, that is, increases in PGC-1a, HO-1, andmitochondrial function not seen in terminally differentiatedadipocytes. The EET-mediated increases in PGC-1a, includ-ing an increase in adiponectin expression and secretion andreduced secretion of proinflammatory cytokines resulting in adecrease in the number of the inflamed adipocyte phenotypes,appear to be dependent on intracellular heme levels as heme isessential for full differentiation of adipocyte [9,57,58]. Theseresults are summarized in Fig. 8. During adipogenesis, MSCsor preadipocytes differentiate into lipid-laden and insulin-sensitive adipocytes [20]. The acquisition of adipocyte phe-notype and development of adipocyte function is characterizedby chronological changes in the expression of multiple genes.Effect of drugs on programmed adipocyte cell differentiationmay influence adipocytes to express adipogenic markers formaturation and terminal differentiation or downregulate suchgenes in favor of an increase in adiponectin and mitochondrialfunction. Adipose tissue and adipocyte cells play an importantrole in insulin resistance through the production and secre-tion of a variety of proteins, including tumor necrosis factor(TNF)-a, IL-6, leptin, and adiponectin [59–62]. In contrast,terminal differentiation is associated with adipocyte enlarge-ment and the expression of several markers, including Peg1/MEST [7,18,50], and proinflammatory cytokines and sup-pression of adiponectin synthesis [18,63]. This process ishighly regulated by the appearance of early, intermediate, and

late mRNA/protein markers and triglyceride accumulation(Fig. 8). This suggests that EET rescues mitochondrial func-tion by activation of PGC-1a and that interplay between EETand PGC-1a is inexorably linked in the regulation of HO-1,heme, and stage of adipocyte growth by modulating the levelof gene expression that leads to adipocyte maturation andterminal differentiation.

Acknowledgments

This work was supported by the National Institutes ofHealth grants HL55601 and HL34300, the Brickstreet Foun-dation and the Huntington Foundation (NGA) and gifts fromthe Ablon, Lang and Renfield Foundations to The RockefellerUniversity (AK). We thank Jennifer Brown for her outstand-ing editorial assistance in the preparation of the article.

Author Disclosure Statement

All authors had full access to the data and take respon-sibility for its integrity. All authors have read and agreedwith the article as written. There are no conflicts of interestamong the authors.

References

1. Zeldin DC. (2001). Epoxygenase pathways of arachidonicacid metabolism. J Biol Chem 276:36059–36062.

2. Capdevila JH, JR Falck, and RC Harris. (2000). Cyto-chrome P450 and arachidonic acid bioactivation. Molecularand functional properties of the arachidonate mono-oxygenase. J Lipid Res 41:163–181.

3. Node K, Y Huo, X Ruan, B Yang, M Spiecker, K Ley, DCZeldin, and JK Liao. (1999). Anti-inflammatory propertiesof cytochrome P450 epoxygenase-derived eicosanoids.Science 285:1276–1279.

4. Spiecker M and JK Liao. (2005). Vascular protective ef-fects of cytochrome p450 epoxygenase-derived eicosa-noids. Arch Biochem Biophys 433:413–420.

5. Vanella L, DH Kim, D Asprinio, SJ Peterson, I Barbagallo,A Vanella, D Goldstein, S Ikehara, A Kappas, and NGAbraham. (2010). HO-1 expression increases mesenchymalstem cell-derived osteoblasts but decreases adipocyte line-age. Bone 46:236–243.

6. Burgess AP, L Vanella, L Bellner, K Gotlinger, JR Falck,NG Abraham, ML Schwartzman, and A Kappas. (2012).Heme oxygenase (HO-1) rescue of adipocyte dysfunctionin HO-2 deficient mice via recruitment of epoxyeicosa-trienoic acids (EETs) and adiponectin. Cell Physiol Bio-chem 29:99–110.

7. Kim DH, L Vanella, K Inoue, A Burgess, K Gotlinger, VLManthati, SR Koduru, DC Zeldin, JR Falck, ML Schwartz-man, and NG Abraham. (2010). Epoxyeicosatrienoic acidagonist regulates human mesenchymal stem cell-derivedadipocytes through activation of HO-1-pAKT signalingand a decrease in PPARgamma. Stem Cells Dev 19:1863–1873.

8. Chen JJ and IM London. (1981). Hemin enhances the differ-entiation of mouse 3T3 cells to adipocytes. Cell 26:117–122.

9. Kumar N, LA Solt, Y Wang, PM Rogers, G Bhattacharyya,TM Kamenecka, KR Stayrook, C Crumbley, ZE Floyd, et al.(2010). Regulation of adipogenesis by natural and syntheticREV-ERB ligands. Endocrinology 151:3015–3025.

1092 WALDMAN ET AL.

10. Abraham NG, JM Junge, and GS Drummond. (2016).Translational significance of heme oxygenase in obesityand metabolic syndrome. Trends Pharmacol Sci 37:17–36.

11. Wu Z, P Puigserver, U Andersson, C Zhang, G Adelmant,V Mootha, A Troy, S Cinti, B Lowell, RC Scarpulla, andBM Spiegelman. (1999). Mechanisms controlling mito-chondrial biogenesis and respiration through the thermo-genic coactivator PGC-1. Cell 98:115–124.

12. Wu N, L Yin, EA Hanniman, S Joshi, and MA Lazar. (2009).Negative feedback maintenance of heme homeostasis by itsreceptor, Rev-erbalpha. Genes Dev 23:2201–2209.

13. Burgess A, L Vanella, L Bellner, ML Schwartzman, andNG Abraham. (2012). Epoxyeicosatrienoic acids and hemeoxygenase-1 interaction attenuates diabetes and metabolicsyndrome complications. Prostaglandins Other Lipid Mediat97:1–16.

14. Li P, JL Lahvic, V Binder, EK Pugach, EB Riley, OJ Tam-plin, D Panigrahy, TV Bowman, FG Barrett, et al. (2015).Epoxyeicosatrienoic acids enhance embryonic haematopoi-esis and adult marrow engraftment. Nature 523:468–471.

15. Lavrovsky Y, ML Schwartzman, RD Levere, A Kappas,and NG Abraham. (1994). Identification of binding sites fortranscription factors NF-kappa B and AP-2 in the promoterregion of the human heme oxygenase 1 gene. Proc NatlAcad Sci U S A 91:5987–5991.

16. Sodhi K, N Puri, K Inoue, JR Falck, ML Schwartzman, andNG Abraham. (2012). EET agonist prevents adiposity andvascular dysfunction in rats fed a high fat diet via a de-crease in Bach 1 and an increase in HO-1 levels. Pros-taglandins Other Lipid Mediat 98:133–142.

17. Zha W, ML Edin, KC Vendrov, RN Schuck, FB Lih, JL Jat,JA Bradbury, LM Degraff, K Hua, et al. (2014). Functionalcharacterization of cytochrome P450-derived epoxyeicosa-trienoic acids in adipogenesis and obesity. J Lipid Res 55:2124–2136.

18. Vanella L, K Sodhi, DH Kim, N Puri, M Maheshwari, TDHinds, Jr., L Bellner, D Goldstein, SJ Peterson, JI Shapiro,and NG Abraham. (2013). Increased heme-oxygenase 1expression decreases adipocyte differentiation and lipidaccumulation in mesenchymal stem cells via upregulationof the canonical Wnt signaling cascade. Stem Cell Res Ther4:28.

19. Vazquez-Vela ME, N Torres, and AR Tovar. (2008). Whiteadipose tissue as endocrine organ and its role in obesity.Arch Med Res 39:715–728.

20. Lefterova MI and MA Lazar. (2009). New developments inadipogenesis. Trends Endocrinol Metab 20:107–114.

21. Rosen ED, CJ Walkey, P Puigserver, and BM Spiegelman.(2000). Transcriptional regulation of adipogenesis. Genes Dev14:1293–1307.

22. Gregoire FM, CM Smas, and HS Sul. (1998). Understandingadipocyte differentiation. Physiol Rev 78:783–809.

23. Burgess A, M Li, L Vanella, DH Kim, R Rezzani, LRodella, K Sodhi, M Canestraro, P Martasek, SJ et al.(2010). Adipocyte heme oxygenase-1 induction attenuatesmetabolic syndrome in both male and female obese mice.Hypertension 56:1124–1130.

24. Ndisang JF. (2010). Role of heme oxygenase in inflam-mation, insulin-signalling, diabetes and obesity. MediatorsInflamm 2010:359732.

25. Peterson SJ, G Drummond, DH Kim, M Li, AL Kruger, SIkehara, and NG Abraham. (2008). L-4F treatment reducesadiposity, increases adiponectin levels, and improves in-sulin sensitivity in obese mice. J Lipid Res 49:1658–1669.

26. Nicolai A, M Li, DH Kim, SJ Peterson, L Vanella, V Po-sitano, A Gastaldelli, R Rezzani, LF Rodella, et al. (2009).Heme oxygenase-1 induction remodels adipose tissue andimproves insulin sensitivity in obesity-induced diabeticrats. Hypertension 53:508–515.

27. Zhang S, G Chen, N Li, M Dai, C Chen, P Wang, H Tang,SL Hoopes, DC Zeldin, DW Wang, and X Xu. (2015).CYP2J2 overexpression ameliorates hyperlipidemia viaincreased fatty acid oxidation mediated by the AMPKpathway. Obesity (Silver Spring) 23:1401–1413.

28. Abraham NG, K Sodhi, AM Silvis, L Vanella, G Favero, RRezzani, C Lee, DC Zeldin, and Schwartzman ML. (2014).CYP2J2 targeting to endothelial cells attenuates adiposity andvascular dysfunction in mice fed a high-fat diet by repro-gramming adipocyte phenotype. Hypertension 64:1352–1361.

29. Campbell WB. (2000). New role for epoxyeicosatrienoicacids as anti-inflammatory mediators. Trends Pharmacol Sci21:125–127.

30. Omata K, K Abe, HL Sheu, K Yoshida, E Tsutsumi, KYoshinaga, NG Abraham, and M Laniado-Schwartzman.(1992). Roles of renal cytochrome P450-dependent arachi-donic acid metabolites in hypertension. Tohoku J Exp Med166:93–106.

31. Puigserver P, Z Wu, CW Park, R Graves, M Wright, andBM Spiegelman. (1998). A cold-inducible coactivator ofnuclear receptors linked to adaptive thermogenesis. Cell 92:829–839.

32. Bostrom P, J Wu, MP Jedrychowski, A Korde, L Ye, JC Lo,KA Rasbach, EA Bostrom, JH Choi, et al. (2012). A PGC1-alpha-dependent myokine that drives brown-fat-like devel-opment of white fat and thermogenesis. Nature 481:463–468.

33. Puigserver P and BM Spiegelman. (2003). Peroxisomeproliferator-activated receptor-gamma coactivator 1 alpha(PGC-1 alpha): transcriptional coactivator and metabolicregulator. Endocr Rev 24:78–90.

34. Wenz T, SG Rossi, RL Rotundo, BM Spiegelman, and CTMoraes. (2009). Increased muscle PGC-1alpha expressionprotects from sarcopenia and metabolic disease during ag-ing. Proc Natl Acad Sci U S A 106:20405–20410.

35. Kleiner S, RJ Mepani, D Laznik, L Ye, MJ Jurczak, FRJornayvaz, JL Estall, BD Chatterjee, GI Shulman, and BMSpiegelman. (2012). Development of insulin resistance inmice lacking PGC-1alpha in adipose tissues. Proc NatlAcad Sci U S A 109:9635–9640.

36. Li R, N Jen, F Yu, and TK Hsiai. (2011). Assessing mi-tochondrial redox status by flow cytometric methods: vas-cular response to fluid shear stress. Curr Protoc CytomChapter 9:Unit 9.37.

37. Infantino V, V Iacobazzi, A Menga, ML Avantaggiati, andF Palmieri. (2014). A key role of the mitochondrial citratecarrier (SLC25A1) in TNFalpha- and IFNgamma-triggeredinflammation. Biochim Biophys Acta 1839:1217–1225.

38. Alhawaj R, D Patel, MR Kelly, D Sun, and MS Wolin. (2015).Heme biosynthesis modulation via delta-aminolevulinic acidadministration attenuates chronic hypoxia-induced pulmo-nary hypertension. Am J Physiol Lung Cell Mol Physiol308:L719–L728.

39. Sambuceti G, S Morbelli, L Vanella, C Kusmic, C Marini, MMassollo, C Augeri, M Corselli, C Ghersi, et al. (2009).Diabetes impairs the vascular recruitment of normal stemcells by oxidant damage; reversed by increases in pAMPK,heme oxygenase-1 and adiponectin. Stem Cells 27:399–407.

40. Pfaffl MW. (2001). A new mathematical model for relativequantification in real-time RT-PCR. Nucleic Acids Res 29:e45.

EET UPSTREAM OF PGC-1a REGULATES ADIPOCYTE DEVELOPMENT 1093

41. St-Pierre J, J Lin, S Krauss, PT Tarr, R Yang, CB Newgard,and BM Spiegelman. (2003). Bioenergetic analysis ofperoxisome proliferator-activated receptor gamma coacti-vators 1alpha and 1beta (PGC-1alpha and PGC-1beta) inmuscle cells. J Biol Chem 278:26597–26603.

42. Baar K, AR Wende, TE Jones, M Marison, LA Nolte, MChen, DP Kelly, and JO Holloszy. (2002). Adaptations ofskeletal muscle to exercise: rapid increase in the tran-scriptional coactivator PGC-1. FASEB J 16:1879–1886.

43. Terada S and I Tabata. (2004). Effects of acute bouts ofrunning and swimming exercise on PGC-1alpha proteinexpression in rat epitrochlearis and soleus muscle. Am JPhysiol Endocrinol Metab 286:E208-E216.

44. H Pilegaard, B Saltin, and PD Neufer. (2003). Exercise in-duces transient transcriptional activation of the PGC-1alphagene in human skeletal muscle. J Physiol 546:851–858.

45. Rhee J, Y Inoue, JC Yoon, P Puigserver, M Fan, FJ Gonzalez,and BM Spiegelman. (2003). Regulation of hepatic fastingresponse by PPARgamma coactivator-1alpha (PGC-1): re-quirement for hepatocyte nuclear factor 4alpha in gluconeo-genesis. Proc Natl Acad Sci U S A 100:4012–4017.

46. Skurk T, C Alberti-Huber, C Herder, and H Hauner. (2007).Relationship between adipocyte size and adipokine ex-pression and secretion. J Clin Endocrinol Metab 92:1023–1033.

47. Drummond GS and A Kappas. (1981). Prevention of neo-natal hyperbilirubinemia by tin protoporphyrin IX, a potentcompetitive inhibitor of heme oxidation. Proc Natl AcadSci U S A 78:6466–6470.

48. Handschin C, J Lin, J Rhee, AK Peyer, S Chin, PH Wu, UAMeyer, and BM Spiegelman. (2005). Nutritional regulationof hepatic heme biosynthesis and porphyria through PGC-1alpha. Cell 122:505–515.

49. Peterson SJ, DH Kim, M Li, V Positano, L Vanella, LFRodella, F Piccolomini, N Puri, A Gastaldelli, C Kusmic,L’Abbate A, and NG Abraham. (2009). The L-4F mimeticpeptide prevents insulin resistance through increased levelsof HO-1, pAMPK, and pAKT in obese mice. J Lipid Res50:1293–1304.

50. Takahashi M, Y Kamei, and O Ezaki. (2005). Mest/Peg1imprinted gene enlarges adipocytes and is a marker of adi-pocyte size. Am J Physiol Endocrinol Metab 288:E117–E124.

51. Vanella L, Li M, D Kim, G Malfa, L Bellner, T Kawakami,and Abraham NG. (2012). ApoA1: mimetic peptide reversesadipocyte dysfunction in vivo and in vitro via an increase inheme oxygenase (HO-1) and Wnt10b. Cell Cycle 11:706–714.

52. Vanella L, DH Kim, K Sodhi, I Barbagallo, AP Burgess, JRFalck, ML Schwartzman, and NG Abraham. (2011). Crosstalkbetween EET and HO-1 downregulates Bach1 and adipogenicmarker expression in mesenchymal stem cell derived adipo-cytes. Prostaglandins Other Lipid Mediat 96:54–62.

53. Abraham NG and A Kappas. (2008). Pharmacological andclinical aspects of heme oxygenase. Pharmacol Rev 60:79–127.

54. Balla J, HS Jacob, G Balla, K Nath, JW Eaton, and GMVercellotti. (1993). Endothelial-cell heme uptake from

heme proteins: induction of sensitization and desensitiza-tion to oxidant damage. Proc Natl Acad Sci U S A 90:9285–9289.

55. Di Noia MA, DS Van, F Palmieri, LM Yang, S Quan, AIGoodman, and NG Abraham. (2006). Heme oxygenase-1enhances renal mitochondrial transport carriers and cyto-chrome C oxidase activity in experimental diabetes. J BiolChem 281:15687–15693.

56. Piantadosi CA, MS Carraway, A Babiker, and HB Suliman.(2008). Heme oxygenase-1 regulates cardiac mitochondrialbiogenesis via Nrf2-mediated transcriptional control ofnuclear respiratory factor-1. Circ Res 103:1232–1240.

57. Chen JK, JR Falck, KM Reddy, J Capdevila, and RCHarris. (1998). Epoxyeicosatrienoic acids and their sulfo-nimide derivatives stimulate tyrosine phosphorylation andinduce mitogenesis in renal epithelial cells. J Biol Chem273:29254–29261.

58. Puri N, K Sodhi, M Haarstad, DH Kim, S Bohinc, E Foglio,G Favero, and NG Abraham. (2012). Heme induced oxi-dative stress attenuates sirtuin1 and enhances adipogenesisin mesenchymal stem cells and mouse pre-adipocytes. JCell Biochem 113:1926–1935.

59. Kershaw EE and JS Flier. (2004). Adipose tissue as anendocrine organ. J Clin Endocrinol Metab 89:2548–2556.

60. Berg AH and PE Scherer. (2005). Adipose tissue, inflam-mation, and cardiovascular disease. Circ Res 96:939–949.

61. Fain JN, Madan AK, Hiler ML, P Cheema, and SW Ba-houth. (2004). Comparison of the release of adipokines byadipose tissue, adipose tissue matrix, and adipocytes fromvisceral and subcutaneous abdominal adipose tissues ofobese humans. Endocrinology 145:2273–2282.

62. Kim JY, van de WE, M Laplante, A Azzara, ME Trujillo,SM Hofmann, T Schraw, JL Durand, H Li, et al. (2007).Obesity-associated improvements in metabolic profile throughexpansion of adipose tissue. J Clin Invest 117:2621–2637.

63. Kim DH, N Puri, K Sodhi, JR Falck, NG Abraham, JShapiro, and ML Schwartzman. (2013). Cyclooxygenase-2dependent metabolism of 20-HETE increases adiposity andadipocyte enlargement in mesenchymal stem cell-derivedadipocytes. J Lipid Res 54:786–793.

Address correspondence to:Dr. Nader G. Abraham

Departments of Medicine and PharmacologyNew York Medical College

15 Dana RoadBasic Science Building, Room 509

Valhalla, NY 10595-1554

E-mail: nader_abraham@nymc.edu

Received for publication March 18, 2016Accepted after revision May 20, 2016

Prepublished on Liebert Instant Online May 25, 2016

1094 WALDMAN ET AL.

This article has been cited by:

1. Francesca Schinzari, Manfredi Tesauro, Carmine Cardillo. 2017. Endothelial and Perivascular Adipose Tissue Abnormalities inObesity-Related Vascular Dysfunction. Journal of Cardiovascular Pharmacology 69:6, 360-368. [CrossRef]

2. Eun-Jeong Koh, Kui-Jin Kim, Young-Jin Seo, Jia Choi, Boo-Yong Lee. 2017. Modulation of HO-1 by Ferulic Acid AttenuatesAdipocyte Differentiation in 3T3-L1 Cells. Molecules 22:5, 745. [CrossRef]

3. Dariusz Galkowski, Mariusz Z. Ratajczak, Janusz Kocki, Zbigniew Darzynkiewicz. 2017. Of Cytometry, Stem Cells and Fountainof Youth. Stem Cell Reviews and Reports 166. . [CrossRef]

4. Mikhail Romashko, Joseph Schragenheim, Nader G. Abraham, John A. McClung. 2016. Epoxyeicosatrienoic Acid as Therapyfor Diabetic and Ischemic Cardiomyopathy. Trends in Pharmacological Sciences 37:11, 945-962. [CrossRef]

5. Shailendra P. Singh, Joseph Schragenheim, Jian Cao, John R. Falck, Nader G. Abraham, Lars Bellner. 2016. PGC-1 alpharegulates HO-1 expression, mitochondrial dynamics and biogenesis: Role of epoxyeicosatrienoic acid. Prostaglandins & Other LipidMediators 125, 8-18. [CrossRef]

6. Shailendra P. Singh, Lars Bellner, Luca Vanella, Jian Cao, John R. Falck, Attallah Kappas, Nader G. Abraham. 2016.Downregulation of PGC-1 α Prevents the Beneficial Effect of EET-Heme Oxygenase-1 on Mitochondrial Integrity and AssociatedMetabolic Function in Obese Mice. Journal of Nutrition and Metabolism 2016, 1-15. [CrossRef]